The present invention relates to broad-spectrum disease resistance in plants, including the phenomenon of systemic acquired resistance (SAR). More particularly, the present invention relates to the identification, isolation and characterization of genes encoding proteins that are involved in the regulation of SAR gene expression in plants.
Plants are constantly challenged by a wide variety of pathogenic organisms including viruses, bacteria, fungi, and nematodes. Crop plants are particularly vulnerable because they are usually grown as genetically-uniform monocultures; when disease strikes, losses can be severe. However, most plants have their own innate mechanisms of defense against pathogenic organisms. Natural variation for resistance to plant pathogens has been identified by plant breeders and pathologists and bred into many crop plants. These natural disease resistance genes often provide high levels of resistance to or immunity against pathogens. Systemic acquired resistance (SAR) is one component of the complex system plants use to defend themselves from pathogens (Hunt and Ryals, 1996; Ryals et al., 1996). See also, U.S. Pat. No. 5,614,395. SAR is a particularly important aspect of plant-pathogen responses because it is a pathogen-inducible, systemic resistance against a broad spectrum of infectious agents, including viruses, bacteria, and fungi. When the SAR signal transduction pathway is blocked, plants become more susceptible to pathogens that normally cause disease, and they also become susceptible to some infectious agents that would not normally cause disease (Gaffney et al, 1993; Delaney et al, 1994; Delaney et al., 1995; Delaney, 1997; Bi et al., 1995; Mauch-Mani and Slusarenko, 1996). These observations indicate that the SAR signal transduction pathway is critical for maintaining plant health.
Conceptually, the SAR response can be divided into two phases. In the initiation phase, a pathogen infection is recognized, and a signal is released that travels through the phloem to distant tissues. This systemic signal is perceived by target cells, which react by expression of both SAR genes and disease resistance. The maintenance phase of SAR refers to the period of time, from weeks up to the entire life of the plant during which the plant is in a quasi steady state, and disease resistance is maintained (Ryals et al., 1996).
Salicylic acid (SA) accumulation appears to be required for SAR signal transduction. Plants that cannot accumulate SA due to treatment with specific inhibitors, epigenetic repression of phenylalanine ammonia-lyase, or transgenic expression of salicylate hydroxylase, which specifically degrades SA, also cannot induce either SAR gene expression or disease resistance (Gaffney et al., 1993; Delaney et al., 1994; Mauch-Mani and Slusaenko, 1996; Maher et al., 1994; Pallas et al., 1996). Although it has been suggested that SA might serve as the systemic signal, this is currently controversial and, to date, all that is known for certain is that if SA cannot accumulate, then SAR signal transduction is blocked (Pallas et al., 1996; Shulaev et a., 1995; Vernooij et al., 1994).
Recently, Arabidopsis has emerged as a model system to study SAR (Uknes et al., 1992; Uknes et a., 1993; Cameron et al., 1994; Mauch-Mani and Slusarenko, 1994; Dempsey and Klessig, 1995). It has been demonstrated that SAR can be activated in Arabidopsis by both pathogens and chemicals, such as SA, 2,6-dichloroisonicotinic acid (INA) and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) (Uknes et al., 1992; Vernooij et al., 1995; Lawton et al, 1996). Following treatment with either INA or BTH or pathogen infection, at least three pathogenesis-related (PR) protein genes, namely, PR-1, PR-2, and PR-5 are coordinately induced concomitant with the onset of resistance (Uknes et al., 1992, 1993). In tobacco, the best characterized species, treatment with a pathogen or an immunization compound induces the expression of at least nine sets of genes (Ward et al., 1991). Transgenic disease-resistant plants have been created by transforming plants with various SAR genes (U.S. Pat. No. 5,614,395).
A number of Arabidopsis mutants have been isolated that have modified SAR signal transduction (Delaney, 1997) The first of these mutants are the so-called lsd (lesions simulating disease) mutants and acd2 (accelerated cell death) (Dietrich et al., 1994; Greenberg et al., 1994). These mutants all have some degree of spontaneous necrotic lesion formation on their leaves, elevated levels of SA, mRNA accumulation for the SAR genes, and significantly enhanced disease resistance. At least seven different lsd mutants have been isolated and characterized (Dietrich et al., 1994; Weymam et al., 1995). Another interesting class of mutants are cim (constitutive immunity) mutants (Lawton et al., 1993). See also, U.S. Pat. No. 5,792,904 and International PCT Application WO 94/16077. Like lsd mutants and acd2, cim mutants have elevated SA and SAR gene expression and resistance, but in contrast to lsd or acd2, do not display detectable lesions on their leaves. cpr1 (constitutive expresser of PR genes) may be a type of cim mutant; however, because the presence of microscopic lesions on the leaves of cpr1 has not been ruled out, cpr1 might be a type of lsd mutant (Bowling et al., 1994).
Mutants have also been isolated that are blocked in SAR signaling ndrl (non-race-specific disease resistance) is a mutant that allows growth of both Pseudomonas syringae containing various avirulence genes and also normally avirulent isolates of Peronospora parasitica (Century et al., 1995). Apparently this mutant is blocked early in SAR signaling. npr1 (nonexpresser of PR genes) is a mutant that cannot induce expression of the SAR signaling pathway following INA treatment (Cao et al., 1994). eds (enhanced disease susceptibility) mutants have been isolated based on their ability to support bacterial infection following inoculation of a low bacterial concentration (Glazebrook et al., 1996; Parker et al., 1996). Certain eds mutants are phenotypically very similar to npr1, and, recently, eds5 and eds53 have been shown to be allelic to npr1 (Glazebrook et al., 1996). nim1 (noninducible immunity) is a mutant that supports P. parasitica (i.e., causal agent of downy mildew disease) growth following INA treatment (Delaney et al., 1995; U.S. Pat. No. 5,792,904). Although nim1 can accumulate SA following pathogen infection, it cannot induce SAR gene expression or disease resistance, suggesting that the mutation blocks the pathway downstream of SA. nim1 is also impaired in its ability to respond to INA or BTH, suggesting that the block exists downstream of the action of these chemicals (Delaney et al., 1995; Lawton et al., 1996).
Allelic Arabidopsis genes have been isolated and characterized, mutants of which are responsible for the nim1 and npr1 phenotypes, respectively (Ryals et al., 1997; Cao et al., 1997). The wild-type NIM1 gene product is involved in the signal transduction cascade leading to both SAR and gene-for-gene disease resistance in Arabidopsis (Ryals et al., 1997). Ryals et al., 1997 also report the isolation of five additional alleles of nim1 that show a range of phenotype from weakly impaired in chemically induced PR-1 gene expression and fungal resistance to very strongly blocked. Transformation of the wild-type NPR1 gene into npr1 mutants not only complemented the mutations, restoring the responsiveness of SAR induction with respect to PR-gene expression and disease resistance, but also rendered the transgenic plants more resistant to infection by P. syringae in the absence of SAR induction (Cao et al., 1997). WO 9806748 describes the isolation of NPR1 from Arabidopsis and a homologue from Nicotiana glutinosa. See also, WO 97/49822, WO 98/26082, and WO 98/29537.
Despite much research and the use of sophisticated and intensive crop protection measures, including genetic transformation of plants, losses due to disease remain in the billions of dollars annually. Therefore, there is a continuing need to develop new crop protection measures based on the ever-increasing understanding of the genetic basis for disease resistance in plants. In particular, there is a long-felt need for the identification, isolation, and characterization of more genes involved in the signal transduction cascade leading to systemic acquired resistance in plants.
The present invention addresses the aforementioned long-felt needs by using the Arabidopsis NIM1 gene as bait in a two-hybrid screen to identify plant proteins that interact with the NIM1 protein. The novel NI16 and NI19 genes are obtainable by this screen. Homologues of the NI16 gene are obtainable by PCR amplification of total cellular DNA from other plants such as potato and tomato. The NI16 gene is rapidly induced following treatment with salicylic acid (SA) and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), as well as by pathogen inoculation. The NI16 gene is also induced in NIM1-overexpressing plants treated with BTH. Transgenic expression of the NI16 gene results in elevated levels of the pathogenesis-related (PR) protein PR-1. The amount of PR-1 induced correlates with the amount of NI16 present, i.e., transgenic plants with the highest expression of NI16 have the highest levels of PR-1. It is believed that NI16 is a necessary component of the SAR response, and that overexpression of NI16 activates a subset of the response. PR-1 is also elevated in nahG and nim1-1 plants overexpressing NI16, which is significant because both nim1 and nahG plants are normally deficient in their ability to induce PR-1 in response to pathogens. Thus, the NI16 gene encodes a protein involved in the regulation of SAR gene expression in plants. The NI16 gene can be expressed in transgenic plants, either alone or in combination with the NIM1 protein, to enhance expression of SAR genes such as PR-1.
In particular, the present invention is directed to an isolated nucleic acid molecule comprising a sequence that encodes the amino acid sequence of SEQ ID NO:2. SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:23.
The present invention is further directed to an isolated nucleic acid molecule comprising the coding region of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:22.
The present invention is also directed to an isolated nucleic acid molecule comprising a sequence that encodes a protein involved in the signal transduction cascade leading to systemic acquired resistance in plants, wherein said sequence comprises a 20, 25, 30,35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion of the coding region of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:22.
The present invention is further directed to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a protein involved in the signal transduction cascade leading to systemic acquired resistance in plants, wherein said nucleotide sequence can be amplified from total cellular DNA from a plant using the polymerase chain reaction with the primers shown as SEQ ID NO:20 and SEQ ID NO:21.
The present invention also encompasses a chimeric gene comprising a promoter active in plants operatively linked to a coding sequence of the present invention, a recombinant vector comprising such a chimeric gene, wherein the vector is capable of being stably transformed into a host, as well as a host stably transformed with such a vector. Preferably, the host is a plant such as one of the following agronomically important crops: rice, wheat, barley, rye, rape, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum, and sugarcane.
In another aspect, the present invention provides the nucleic acid sequence of the NI16 promoter, an example of which is presented in SEQ ID NO:3. A preferred embodiment of the NI16 promoter comprises the region upstream of nucleotide number 863 of SEQ ID NO:3. In addition, the present invention encompasses chimeric genes comprising the NI16 promoter operatively linked to a coding sequence of a gene of interest, wherein the NI16 promoter regulates transcription of the coding sequence in the presence of chemical regulators. In a preferred embodiment, the coding sequence encodes an enzyme, such as an assayable marker, whereby expression of the marker can be observed in assays for chemical induction of the chimeric gene. In related aspects, the present invention also embodies a recombinant vector, such as a plasmid, comprising the aforementioned chimeric gene, as well as a plant or plant tissue stably transformed with such a chimeric gene.
SEQ ID NO:1xe2x80x94Full length cDNA sequence of the Arabidopsis thaliana NI16 gene.
SEQ ID NO:2xe2x80x94Amino acid sequence of the NI16 protein encoded by SEQ ID NO:1.
SEQ ID NO:3xe2x80x94Genomic DNA sequence of the Arabidopsis thaliana NI16 gene region, including the 5xe2x80x2 upstream promoter sequence.
SEQ ID NO:4xe2x80x94Full length cDNA sequence of the potato NI16 homologue.
SEQ ID NO:5xe2x80x94Potato NI16 amino acid sequence encoded by SEQ ID NO:4.
SEQ ID NO:6xe2x80x94Partial cDNA sequence of the tomato NI16 homologue.
SEQ ID NO:7xe2x80x94Tomato NI16 amino acid sequence encoded by SEQ ID NO:6.
SEQ ID NO:8xe2x80x94Amino acid sequence encoded by soybean EST AI495 102.
SEQ ID NO:9xe2x80x94Amino acid sequence encoded by soybean EST AI461039.
SEQ ID NO:10xe2x80x94Amino acid sequence encoded by tobacco G8-1 gene.
SEQ ID NO:11xe2x80x94Primer NIM5xe2x80x2RI
SEQ ID NO:12xe2x80x94Primer NIM3xe2x80x2SalI
SEQ ID NO:13xe2x80x94Primer NIMtrunc3xe2x80x2NcoI
SEQ ID NO:14xe2x80x94Primer NIMloop5xe2x80x2RI
SEQ ID NO:15xe2x80x94Primer 16GSP1
SEQ ID NO:16xe2x80x94Primer 16GSP2
SEQ ID NO:17xe2x80x94Primer 16GSP3
SEQ ID NO:18xe2x80x94Primer 16F
SEQ ID NO:19xe2x80x94Primer 16R
SEQ ID NO:20xe2x80x94Primer NI16-DegF
SEQ ID NO:21xe2x80x94Primer NI16-DegR
SEQ ID NO:22xe2x80x94Genomic sequence of the Arabidopsis thaliana NI19 gene.
SEQ ID NO:23xe2x80x94Amino acid sequence of the NI19 protein encoded by SEQ ID NO:22.
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
Associated With/Operatively Linked: Refers to two DNA sequences that are related physically or functionally. For example, a promoter or regulatory DNA sequence is said to be xe2x80x9cassociated withxe2x80x9d a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.
Chimeric Gene: A recombinant DNA sequence in which a promoter or regulatory DNA sequence is operatively linked to, or associated with, a DNA sequence that codes for an mRNA or which is expressed as a protein, such that the regulator DNA sequence is able to regulate transcription or expression of the associated DNA sequence. The regulator DNA sequence of the chimeric gene is not normally operatively linked to the associated DNA sequence as found in nature.
Coding Sequence: a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism to produce a protein.
Complementary: refers to two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between the complementary base residues in the antiparallel nucleotide sequences.
Expression: refers to the transcription and/or translation of an endogenous gene or a transgene in plants. In the case of antisense constructs, for example, expression may refer to the transcription of the antisense DNA only.
Expression Cassette: A nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue, or organ, or stage of development.
Gene: A defined region that is located within a genome and that, besides the aforementioned coding nucleic acid sequence, comprises other, primarily regulatory, nucleic acid sequences responsible for the control of expression, i.e., transcription and translation of the coding portion. A gene may also comprise other 5xe2x80x2 and 3xe2x80x2 untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.
Heterologous DNA Sequence: The terms xe2x80x9cheterologous DNA sequencexe2x80x9d, xe2x80x9cexogenous DNA segmentxe2x80x9d or xe2x80x9cheterologous nucleic acid,xe2x80x9d as used herein, each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
Homologous DNA Sequence: A DNA sequence naturally associated with a host cell into which it is introduced.
Isocoding: A nucleic acid sequence is isocoding with a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.
Isolated: In the context of the present invention, an isolated nucleic acid molecule or an isolated enzyme is a nucleic acid molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell.
Minimal Promoter: a promoter element, particularly a TATA element, that is inactive or has greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, a minimal promoter functions to permit transcription.
Native: refers to a gene that is present in the genome of an untransformed cell.
Naturally occurring: the term xe2x80x9cnaturally occurringxe2x80x9d is used to describe an object that can be found in nature as distinct from being artificially produced by man. For example, a protein or nucleotide sequence it present in an organism (including a virus), which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.
NI16: Gene involved in the SAR signal transduction cascade that interacts with the NIM1 protein.
NIM1: Gene described in Ryals et al., 1997, which is involved in the SAR signal transduction cascade.
NIM1: Protein encoded by the NIM1 gene.
Nucleic acid: the term xe2x80x9cnucleic acidxe2x80x9d refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)). The terms xe2x80x9cnucleic acidxe2x80x9d or xe2x80x9cnucleic acid sequencexe2x80x9d may also be used interchangeably with gene, cDNA, and mRNA encoded by a gene. In the context of the present invention, the nucleic acid molecule is preferably a segment of DNA. Nucleotides are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G).
ORF: Open Reading Frame.
Plant: Any whole plant.
Plant Cell: Structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized Unit such as, for example, a plant tissue, a plant organ, or a whole plant.
Plant Cell Culture: Cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
Plant Material: Refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
Plant Organ: A distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
Plant tissue: A group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
Promoter: An untranslated DNA sequence upstream of the coding region that contains the binding site for RNA polymerase II and initiates transcription of the DNA. The promoter region may also include other elements that act as regulators of gene expression.
Protoplast: An isolated plant cell without a cell wall or with only parts of the cell wall.
Purified: the term xe2x80x9cpurified,xe2x80x9d when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified. The term xe2x80x9cpurifiedxe2x80x9d denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least about 50% pure, more preferably at least about 85% pure, and most preferably at least about 99% pure.
Recombinant DNA molecule: a combination of DNA molecules teat are joined together using recombinant DNA technology
Regulatory Elements: Sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.
Selectable marker gene: a gene whose expression in a plant cell gives the cell a selective advantage. The selective advantage possessed by the cells transformed with the selectable marker gene may be due to their ability to grow in the presence of a negative selective agent, such as an antibiotic or a herbicide, compared to the growth of non-transformed cells. The selective advantage possessed by the transformed cells, compared to non-transformed cells, may also be due to their enhanced or novel capacity to utilize an added compound as a nutrient, growth factor or energy source. Selectable marker gene also refers to a gene or a combination of genes whose expression in a plant cell gives the cell both, a negative and a positive selective advantage.
Significant Increase: an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, more preferably an increase by about 5-fold or greater, and most preferably an increase by about 10-fold or greater.
The terms xe2x80x9cidenticalxe2x80x9d or percent xe2x80x9cidentityxe2x80x9d in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
Substantially identical: the phrase xe2x80x9csubstantially identical,xe2x80x9d in the context of two nucleic acid or protein sequences, refers to two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 90-95%, and most preferably at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions. Furthermore, substantially identical nucleic acid or protein sequences perform substantially the same function.
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat""l. Acad Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra).
One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always  greater than 0) and N (penalty score for mismatching residues; always  less than 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad Sci. USA 89: 10915 (1989)).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. Karlin and Altschul, Proc. Nat""l. Acad. Sci., USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase xe2x80x9chybridizing specifically toxe2x80x9d refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. xe2x80x9cBind(s) substantiallyxe2x80x9d refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
xe2x80x9cStringent hybridization conditionsxe2x80x9d and xe2x80x9cstringent hybridization wash conditionsxe2x80x9d in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part 1 chapter 2 xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid probe assaysxe2x80x9d Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Typically, under xe2x80x9cstringent conditionsxe2x80x9d a probe will hybridize to its target subsequence, but to no other sequences.
The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42xc2x0 C., with the hybridization being carried out overnight An example of highly stringent wash conditions is 0.1 5M NaCl at 72xc2x0 C. for about 15 minutes. An example of stringent wash conditions is a 0.2xc3x97SSC wash at 65xc2x0 C. for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1xc3x97SSC at 45xc2x0 C. for 15 minutes. An example low stringency wash for a duplex of e.g., more than 100 nucleotides, is 4-6xc3x97SSC at 40xc2x0 C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30xc2x0 C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2xc3x97 (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
The following are examples of sets of hybridization/wash conditions that may be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NAPO4, 1 mM EDTA at 50xc2x0 C. with washing in 2xc3x97SSC, 0.1% SDS at 50xc2x0 C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NAPO4, 1 mM EDTA at 50xc2x0 C. with washing in 1xc3x97SSC, 0.1% SDS at 50xc2x0 C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50xc2x0 C. with washing in 0.5xc3x97SSC, 0.1% SDS at 50xc2x0 C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50xc2x0 C. with washing in 0.1xc3x97SSC, 0.1% SDS at 50xc2x0 C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NAPO, 1 mM EDTA at 50xc2x0 C. with washing in 0.1xc3x97SSC, 0.1% SDS at 65xc2x0 C.
A further indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions.
The phrase xe2x80x9cspecifically (or selectively) binds to an antibody,xe2x80x9d or xe2x80x9cspecifically (or selectively) immunoreactive with,xe2x80x9d when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to the protein with the amino acid sequence encoded by any of the nucleic acid sequences of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins except for polymorphic variants. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York xe2x80x9cHarlow and Lanexe2x80x9d), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
xe2x80x9cConservatively modified variationsxe2x80x9d of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are xe2x80x9csilent variationsxe2x80x9d which are one species of xe2x80x9cconservatively modified variations.xe2x80x9d Every nucleic acid sequence described herein which encodes a protein also describes every possible silent variation, except where otherwise noted. One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each xe2x80x9csilent variationxe2x80x9d of a nucleic acid which encodes a protein is implicit in each described sequence.
Furthermore, one of skill will recognize that individual substitutions deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are xe2x80x9cconservatively modified variations,xe2x80x9d where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another. Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E),
Asparagine (N), Glutamine (Q). See also, Creighton (1984) Proteins, W.H. Freeman and Company. In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also xe2x80x9cconservatively modified variations.xe2x80x9d
A xe2x80x9csubsequencexe2x80x9d refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e.g., protein) respectively.
Nucleic acids are xe2x80x9celongatedxe2x80x9d when additional nucleotides (or other analogous molecules) are incorporated into the nucleic acid. Most commonly, this is performed with a polymerase (e.g., a DNA polymerase), e.g., a polymerase which adds sequences at the 3xe2x80x2 terminus of the nucleic acid.
Two nucleic acids are xe2x80x9crecombinedxe2x80x9d when sequences from each of the two nucleic acids are combined in a progeny nucleic acid. Two sequences are xe2x80x9cdirectlyxe2x80x9d recombined when both of the nucleic acids are substrates for recombination. Two sequences are xe2x80x9cindirectly recombinedxe2x80x9dwhen the sequences are recombined using an intermediate such as a cross-over oligonucleotide. For indirect recombination, no more than one of the sequences is an actual ,substrate for recombination, and in some cases, neither sequence is a substrate for recombination.
A xe2x80x9cspecific binding affinityxe2x80x9d between two molecules, for example, a ligand and a receptor, means a preferential binding of one molecule for another in a mixture of molecules. The binding of the molecules can be considered specific if the binding affinity is about 1xc3x97104 Mxe2x88x921 to about 1xc3x97106 Mxe2x88x921 or greater.
Transformation: a process for introducing heterologous DNA into a host cell or organism. xe2x80x9c
Transformed,xe2x80x9d xe2x80x9ctransgenic,xe2x80x9d and xe2x80x9crecombinantxe2x80x9d refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be autoreplicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A xe2x80x9cnon-transformed,xe2x80x9d xe2x80x9cnon-transgenic,xe2x80x9d or xe2x80x9cnon-recombinantxe2x80x9d host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
The following material has been deposited with the Agricultural Research Service, Patent Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, USA, under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. All restrictions on the availability of the deposited material will be irrevocably removed upon the granting of a patent.
The Arabidopsis NIM1 gene (Ryals et al., 1997) is used as a bait in a yeast two-hybrid screen to isolate the novel NIM1 interactors NI16 and NI19. The NI16 gene is rapidly induced following treatment with salicylic acid (SA) and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), as well as by pathogen inoculation. The NI16 gene is also induced in NIM1-overexpressing plants treated with BTH. The NI16 gene sequence contains regions of high homology to the tobacco G8-1 gene, which is induced rapidly following treatment with salicylic acid, and which is responsive to very low concentrations of salicylate.
A full length Arabidopsis NI16 cDNA is determined by 5xe2x80x2RACE kit and encodes a protein of 122 amino acids. In addition, a genomic copy of Arabidopsis NI16 is cloned from a genomic library. The genomic clone also contains the NI16 5xe2x80x2 upstream promoter region, which includes several promoter elements in the region adjacent to the NI16 genomic coding region. These promoter elements include: a tandem CaMV AS1 motif, which is known to be SA inducible (Terzaghi and Cashmore, 1995); a tandem TCA1 motif, which is another common SA inducible element (Goldsbrough et al., 1993); a MYCATR22 element, which is a binding site for MCY (rd22BPI) in Arabidopsis dehydration-responsive gene rd22 (ABA-induction); a PAL BOX, which is one of three putative cis-acting elements of the phenylalanine ammonia-lyase family in parsley; and a HEXAMERAT4 element, which is a hexamer motif of the Arabidopsis histone H4 promoter. Because the NI16 gene is rapidly induced following treatment with the SAR inducers SA and BTH, it is predicted that the NI16 promoter is inducible by a full range of known SAR-inducers and can therefore be used for the same purposes as described for the tobacco PR-1a promoter in U.S. Pat. No. 5,614,395 and for the Arabidopsis PR-1 promoter in WO 98/03536.
NI16 and NI19 gene sequences according to the invention can be isolated using the techniques described in the examples below, or by PCR using the NI16 sequences (SEQ ID NO:1 or SEQ ID NO:3) or NI19 sequence (SEQ ID NO:22) set forth in the sequence listing as the basis for constructing PCR primers. For example, oligonucleotides corresponding to the sequence of approximately the first and last 20-25 consecutive nucleotides of the NI16 coding sequence or their complements (e.g., SEQ ID NO:20 and SEQ ID NO:21) can be used as PCR primers to amplify a NI16 gene sequence directly from a cDNA or genomic library from a source plant such as Arabidopsis thaliana, potato or tomato. Thus, the NI16 and NI19 gene sequences set forth in the sequence listing, as well as homologous NI16 and NI19 gene sequences from additional plants, can likewise be amplified by PCR from cDNA and genomic libraries.
Transgenic expression of the NI16 gene results in constitutive expression of PR-1. Hence, the NI16 gene encodes a protein involved in the regulation of SAR gene expression.
A NI16 or N19 coding sequence of the present invention may be inserted into an expression cassette designed for plants to construct a chimeric gene according to the invention using standard genetic engineering techniques. The choice of specific regulatory sequences such as promoter, signal sequence, 5xe2x80x2 and 3xe2x80x2 untranslated sequences, and enhancer appropriate for the achieving the desired pattern and level of expression in the chosen plant host is within the level of skill of the routineer in the art. The resultant molecule, containing the individual elements linked in proper reading frame, may be inserted into a vector capable of being transformed into a host plant cell.
Examples of promoters capable of functioning in plants or plant cells (i.e., those capable of driving expression of associated coding sequences such as those coding for the NI16 or NI19 protein in plant cells) include the Arabidopsis and maize ubiquitin promoters; cauliflower mosaic virus (CAMV) 19S or 35S promoters and CaMV double promoters; rice actin promoters; PR-1 promoters from tobacco, Arabidopsis, or maize; nopaline synthase promoters; small subunit of ribulose bisphosphate carboxylase (ssuRUBISCO) promoters, and the like. Especially preferred is the Arabidopsis ubiquitin promoter. The promoters themselves may be modified to manipulate promoter strength to increase expression of the associated coding sequence in accordance with art-recognized procedures. Preferred promoters for use with the present invention are those that confer high level constitutive expression.
Signal or transit peptides may be fused to the NI16 coding sequence in the chimeric DNA constructs of the invention to direct transport of the expressed protein to the desired site of action. Examples of signal peptides include those natively linked to the plant pathogenesis-related proteins, e.g, PR-1, PR-2, and the like. See, e.g., Payne et al., 1988. Examples of transit peptides include the chloroplast transit peptides such as those described in Von Heijne et al (1991), Mazur et al. (1987), and Vorst et al. (1988); and mitochondrial transit peptides such as those described in Boutry et al (1987). Also included are sequences that result in localization of the encoded protein to various cellular compartments such as the vacuole. See, for example, Neuhaus et al. (1991) and Chrispeels (1991).
The chimeric DNA construct(s) of the invention may contain multiple copies of a promoter or multiple copies of a NI16 or NI19 coding sequence of the present invention. In addition, the constuct(s) may include coding sequences for markers and coding sequences for other peptides such as signal or transit peptides, each in proper reading frame with the other functional elements in the DNA molecule. The preparation of such constructs are within the ordinary level of skill in the art.
Useful markers include peptides providing herbicide, antibiotic or drug resistance, such as, for example, resistance to protoporphyrinogen oxidase inhibitors, hygromycin, kanamycin, G418, gentamycin, lincomycin, methotrexate, glyphosate, phosphinothricin, or the like. These markers can be used to select cells transformed with the chimeric DNA constructs of the invention from untransformed cells. Other useful markers are peptidic enzymes which can be easily detected by a visible reaction, for example a color reaction, for example luciferase, xcex2-glucuronidase, or xcex2-galactosidase.
Chimeric genes designed for plant expression such as those described herein can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant (i.e. monocot or dicot) and/or organelle (i.e. nucleus, chloroplast, mitochondria) targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al., 1986), electroporation (Riggs et al., 1986), Agrobacterium mediated transformation (Hinchee et al., 1988; Ishida et al., 1996), direct gene transfer (Paszkowski et al., 1984; Hayashimoto et al., 1990), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del. (see, for example, U.S. Pat. No. 4,945,050; and McCabe et al., 1988). See also, Weissinger et al. (1988); Sanford et al. (1987) (onion); Christou et al. (1988) (soybean); McCabe et al (1988) (soybean); Datta et al. (1990) (rice); Klein et al. (1988) (maize); Klein et al. (1988) (maize); Klein et al. (1988) (maize); Fromm et al. (1999); and Gordon-Kamm et al. (1990) (maize); Svab et al. (1990) (tobacco chloroplasts); Gordon-Kamm et al. (1993) (maize); Shimamoto et al. (1989) (rice); Christou et al. (1991) (rice), Datta et al. (1990) (rice); European Patent Application EP 0 332 581 (orchardgrass and other Pooideae); Vasil et al. (1993) (wheat); Weeks et al. (1993) (wheat); Wan et al. (1994) (barley); Jahne et al. (1994) (barley); Umbeck et al. (1987) (cotton); Casas et al. (1 993) (sorghum); Somers et al. (1 992) (oats); Torbert et al. (1995) (oats); Weeks et al., (1993) (wheat); WO 94113822 (wheat); and Nehrra et al. (1994) (wheat). A particularly preferred set of embodiments for the introduction of recombinant DNA molecules into maize by microprojectile, bombardment can be found in Koziel et al. (1993); Hill et al. (1995) and Koziel et al. (1996. An additional preferred embodiment is the protoplast transformation method for maize as disclosed in EP 0 292 435.
Once a chimeric gene comprising a NI16 or NI19 coding sequence has been transformed into a particular plant species, it may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques. Preferred plants of the invention include gymnosperms, monocots, and dicots, especially agronomically important crop plants, such as rice, wheat, barley, rye, rape, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane. The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction and can thus be maintained and propagated in progeny plants.